Recombinant Edwardsiella ictaluri ATP synthase subunit c (atpE)

What is the structure and function of Edwardsiella ictaluri ATP synthase subunit c (atpE)?

Edwardsiella ictaluri ATP synthase subunit c is a critical component of the F-type ATPase in this fish pathogen. The protein consists of 79 amino acids with the sequence: MENLSMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA . Functionally, atpE is part of the FO domain of ATP synthase, forming the membrane proton channel that works in conjunction with the F1 domain (the extramembranous catalytic core) . Together, these domains catalyze the production of ATP from ADP in the presence of sodium or proton gradient, a process essential for bacterial energy metabolism . The protein is also known by several alternative names including ATP synthase F(0) sector subunit c, F-type ATPase subunit c, F-ATPase subunit c, and Lipid-binding protein .

How does E. ictaluri atpE differ from ATP synthase subunit c in other pathogenic bacteria?

While ATP synthase subunit c is relatively conserved across bacterial species, significant differences exist in amino acid composition that can affect functionality and drug targeting. E. ictaluri atpE functions optimally at lower temperatures (25-30°C) corresponding to the bacterium's growth conditions, unlike human pathogens that typically function at 37°C . This temperature adaptation reflects E. ictaluri's evolution as a fish pathogen that causes enteric septicemia of catfish (ESC), a primary cause of mortality in aquaculture settings .

The FO domain where atpE resides consists of residues between 5-25 and 57-77, which form the central rotor element of the F1 complex . This structural arrangement shows subtle differences from human ATP synthase, making it a potentially attractive target for antimicrobial development with reduced host toxicity .

What are the optimal storage and handling conditions for recombinant E. ictaluri atpE?

For research applications requiring preserved protein activity, recombinant E. ictaluri atpE should be stored at -20°C in Tris-based buffer with 50% glycerol . For extended storage periods, maintaining samples at -80°C is recommended . Working aliquots can be stored at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided as they may compromise protein integrity and activity .

When working with this protein, researchers should consider E. ictaluri's environmental preferences: it grows optimally at 28-30°C and shows poor growth at 37°C, suggesting that experimental assays should be conducted within this optimal temperature range .

What expression systems are most effective for producing recombinant E. ictaluri atpE?

The expression of functional recombinant E. ictaluri atpE requires careful consideration of the expression system. Based on the protein's characteristics, prokaryotic expression systems using E. coli strains optimized for membrane protein expression are generally suitable. When designing expression constructs, researchers should note that the full-length protein encompasses region 1-79 of the coding sequence, as identified in strain 93-146 (Uniprot accession C5BF35) .

The choice of tag can significantly impact protein folding and activity. While specific tag information for commercial recombinant atpE is typically determined during the production process, His-tags are commonly used for purification of ATP synthase components . Researchers should test multiple tagging strategies (N-terminal vs. C-terminal) to determine which preserves enzymatic function.

How can recombinant E. ictaluri atpE be used in studying bacterial pathogenesis mechanisms?

Recombinant E. ictaluri atpE serves as a valuable tool for investigating energy metabolism during pathogenesis. E. ictaluri is a facultative intracellular pathogen that requires a type III secretion system for survival and replication within channel catfish head kidney-derived macrophages (HKDM) . Researchers can use purified recombinant atpE in competitive binding assays to identify host interactions that may contribute to intracellular survival.

Methodologically, investigators should design experiments that mimic intracellular conditions. E. ictaluri demonstrates significant tolerance to acidic conditions (pH 3.0), which may be relevant to survival in host phagosomes . When studying atpE function during pathogenesis, researchers should incorporate pH conditions that reflect the intracellular environment of fish macrophages, potentially in conjunction with urea supplementation, as E. ictaluri possesses a urease pathogenicity island that may work synergistically with ATP synthase under stress conditions .

What methodological approaches are most effective for studying atpE function in the context of E. ictaluri acid tolerance?

E. ictaluri's ability to survive in acidic environments is a significant virulence factor. While urease activity has been identified as contributing to acid tolerance, the potential role of ATP synthase in this process warrants investigation . To study this relationship, researchers should consider:

• Comparing wild-type and atpE-mutant strains for survival in acidic conditions

• Measuring ATP production under various pH conditions (pH 3.0-7.0)

• Monitoring membrane potential during acid exposure using fluorescent probes

• Conducting gene expression analysis of atpE and related energy metabolism genes during acid stress

Experimental protocols should include appropriate controls such as the ureG::kan urease mutant strain previously described in literature, which allows researchers to differentiate between urease-dependent and ATP synthase-dependent acid tolerance mechanisms .

How can structural analysis of recombinant atpE inform drug development against E. ictaluri?

ATP synthase subunit c represents a promising drug target due to its essential role in bacterial metabolism. Structural characterization of E. ictaluri atpE can guide rational drug design approaches. Researchers have identified compounds that bind to ATP synthase subunit c in other bacterial species with binding energies ranging from -8.69 to -8.44 kcal/mol, demonstrating higher affinity than the natural substrate ATP .

When designing inhibitor screening assays, researchers should consider the structural domains of ATP synthase. The FO domain containing atpE includes residues between 5-25 and 57-77, which should be the primary focus of binding studies .

What techniques can be used to study the interaction between E. ictaluri atpE and bacteriophages?

Bacteriophages specific to E. ictaluri represent both research tools and potential therapeutic agents for controlling ESC in aquaculture . Understanding how these phages interact with bacterial surface proteins, including potentially exposed portions of membrane-embedded proteins like atpE, may inform phage therapy development.

Researchers can employ the following methodological approaches:

• Phage adsorption assays comparing wild-type and atpE-modified E. ictaluri

• Phage resistance development studies in the presence of atpE inhibitors

• Structural analysis of phage-atpE interactions using cryo-electron microscopy

• Comparative genomic analysis of bacteriophages with differential binding to E. ictaluri strains

Recent genomic analysis of E. ictaluri-specific bacteriophages (eiAU, eiDWF, and eiMSLS) revealed genomes of approximately 42 kbp with significant variability in tail fiber proteins, which are responsible for host recognition . These phages, isolated from geographically distinct aquaculture ponds, provide valuable resources for studying phage-host interactions involving membrane proteins like atpE.

How does the expression and function of atpE change under different environmental conditions relevant to fish pathogenesis?

The expression and activity of E. ictaluri atpE likely varies under different environmental conditions encountered during infection. E. ictaluri is known to be weakly motile at 25-30°C but not at higher temperatures, suggesting temperature-dependent regulation of energy metabolism . Additionally, the bacterium can survive at pH 3.0 but cannot grow at pH 5.0 without urea supplementation .

To study these adaptations, researchers should:

• Perform quantitative PCR analysis of atpE expression under varying temperature, pH, and nutrient conditions

• Measure ATP synthesis activity in membrane preparations from bacteria grown under different conditions

• Use fluorescently-labeled recombinant atpE to track protein localization during environmental transitions

• Conduct comparative proteomic analysis to identify post-translational modifications of atpE under stress conditions

These approaches will help elucidate how E. ictaluri modulates energy metabolism during host infection, potentially revealing new targets for therapeutic intervention.